This invention relates to a method for the culture of cells and, more particularly, to the submerged culture of animal cells using microcarriers for the attachment of cells in suspension.
The successful use of microcarriers for cell culture was first reported by van Wezel, Nature 216, 64-65 (1967). The method of van Wezel consisted of growing cells as monolayers on the surface of positively charged DEAE-Sephadex.RTM. beads(grade A-50, about 100.mu. diameter) suspended in culture media in a stirred vessel. The stirred vessel used by van Wezel was the Bilthoven microbial culture unit described by van Hemert, Biotechnol. Bioeng. VI, 381-401 (1964). In this method, different cell lines, human diploid cells, and primary rabbit kidney cells were successfully cultivated. The production of polio virus in the microcarrier culture was examined by van Wezel and the virus multiplication was found to be essentially similar to that in monolayer culture.
Other microcarriers or particulate support materials which have been disclosed heretofore as useful for the attachment of cells in suspension culture are the porous Spherosil.RTM. silica spherules (about 125-150.mu. diameter) described in U.S. Pat. No. 3,717,551; the collodion (nitrocellulose) coated DEAE-Sephadex beads of van Hemert et al, Biotechnol. Bioeng. XI, 875-85 (1969); the glass beads (3.5-4 mm diameter) taught by Wohler et al, Exper. Cell. Res. 74, 571-3 (1972) and German Offenlegungsshrift No. 2,300,567; the QAE-Sephadex, CM-Sephadex and Dowex.RTM. 1-X8 beads described by Horng and McLimans, Biotechnol. Bioeng. XVII, 713-32 (1975); the silicone-treated polycarbonate beads mentioned in Netherlands Patent Application No. 76 05,438, Nov. 23, 1976; the polyacrylonitrile particles taught in U.S. Pat. No. 4,024,020; the CMC-coated DEAE-Sephadex beads disclosed in U.S. Pat. No. 4,036,693; and the formal or butyral coated DEAE-Sephadex beads described in German Offenlegungsshrift No. 2,834,067.
In another prior disclosure, U.S. Pat. No. 3,887,430, certain basic anion exchange resins accompanied by a lipid source such as polysorbate 60 are suggested as useful support materials for the growth of cells in tissue culture. The resins used in said combination with a lipid source are described as based on a vinyl aromatic compound such as styrene copolymerized with a cross-linking agent such as divinylbenzene and then reacted with a halomethylating agent such as chloromethyl methyl ether to produce a halomethylated polystyrene. The halomethylated polymeric product is then reacted with a primary or secondary amine or with a polyamine such as polyalkylenepolyamine to produce the basic anion exchange resin. These resins are stated to be useful for tissue culture of animal cells such as primary chick embryo and Madin-Darby bovine kidney cells and for propagating virus such as measles and mumps virus.
Notwithstanding the variety of microcarriers taught in foregoing art, the discovery and development of new types of microcarriers for cell culture is much sought after since the particular requirements for culture of certain cells are not satisfactorily met by the known microcarriers.